Wide-field three-photon excitation in biological samples

Abstract

Three-photon wide-field depth-resolved excitation is used to overcome some of the limitations in conventional point-scanning two- and three-photon microscopy. Excitation of chromophores as diverse as channelrhodopsins and quantum dots is shown, and a penetration depth of more than 700 μm into fixed scattering brain tissue is achieved, approximately twice as deep as that achieved using two-photon wide-field excitation. Compatibility with live animal experiments is confirmed by imaging the cerebral vasculature of an anesthetized mouse; a complete focal stack was obtained without any evidence of photodamage. As an additional validation of the utility of wide-field three-photon excitation, functional excitation is demonstrated by performing three-photon optogenetic stimulation of cultured mouse hippocampal neurons expressing a channelrhodopsin; action potentials could reliably be excited without causing photodamage.

Two‐ and three‐photon absorption cross‐section characterization for high‐brightness, cell‐specific multiphoton fluorescence brain imaging

“…As a general tendency, seen from the 2PEF and 3PEF images in 3AD, 3PEF images are much sharper and show a much higher contrast compared to their 2PEF counterparts. This finding agrees well with the earlier three‐photon imaging studies .…”

“…In their seminal work, Horton et al have shown that microscopy based on three‐photon‐excited fluorescence (3PEF) enables a noninvasive, high‐resolution in vivo imaging of subcortical structures within an intact mouse brain. Significant recent milestones in the development of 3PEF imaging technology include demonstration of optical imaging of neuronal activity deep in intact mouse brain , wide‐field three‐photon optogenetic stimulation , brain structure and function detection through an intact skull , and 3PEF light‐sheet microscopy .…”

Quantitative Analysis of 1300-nm Three-photon Calcium Imaging in the Mouse Brain

“…Since the first demonstrations of 3PM for in vivo brain imaging (Horton et al, 2013; Ouzounov et al, 2017), a number of research groups have successfully adopted and developed the technology (Chen et al, 2018; Escobet-Montalbán et al, 2018; Odríguez, 2018; Perillo et al, 2017; Rowlands et al, 2017; Takasaki et al, 2019b; Weisenburger et al, 2019; Yildirim et al, 2019), propelled by the commercially available lasers and microscopes. To facilitate 3PM applications, here we provide a quantitative characterization of three-photon deep-brain calcium imaging, with a side-by-side comparison to 2PM with 920 nm excitation.…”

Wide-field multiphoton imaging with TRAFIX

“…Similarly to other multiphoton imaging techniques, TRAFIX can be extended to higher order multiphoton processes, such as 3P excitation, to potentially achieve better penetration depth. 23,26 3P microscopy usually relies on long wavelengths (1300 nm and 1700 nm), which are more resistant to scattering and achieve deeper penetration inside biological tissue. It has also been shown that by relying on such higher order nonlinear process, fluorescence is confined to a smaller volume, reducing out-of-focus light.…”

“…pulsed lasers typically have pulse duration and repetition rate below 70 fs and 1.25 MHz, [17][18][19][20][21]23 respectively. The Ti:Sapphire laser source in our system is typically used to generate 2P excitation, therefore, it is not optimal for 3P microscopy.…”